Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include sporadic Creutzfeldt-Jakob disease (sCJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in mule deer and elk. Prions can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding prion pathogenesis and developing effective therapeutics. The infectious agent of prion diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrPSc) of the normal, protease-sensitive prion protein, PrPC. Susceptibility to infection can be influenced by amino acid homology between PrPC and PrPSc while differences in structure between PrPSc molecules are believed to encode strain phenotypes. My laboratory addresses many different aspects of prion diseases at both the molecular and pathogenic level including: 1) identifying the earliest events which occur during prion infection, 2) defining the molecular pathways involved in prion-associated neurodegeneration, 3) determining the molecular basis of prion strains, 4) determining how PrPC sequence and post-translational modifications influence PrPSc formation and disease phenotype and, 5) development of effective prion therapeutics. Although there is an increasing body of work suggesting that mitochondrial dysfunction is important in several neurodegenerative protein misfolding diseases such as Alzheimers disease (AD) and Parkinsons disease (PD), the role of mitochondria in prion disease is poorly understood. We recently published data showing that PrPC is present in brain mitochondria from healthy wild-type and transgenic mice (Faris et al., Sci. Rep. 7: 41556 (2017), Annual Report 2017). We have also found evidence that mitochondria may be impaired in mice overexpressing PrPC. (Faris et al., J. Virol. 91: e00524-17 (2017), Annual Report 2017). Our data suggest that, as has been proposed for other proteins associated with neurodegenerative disorders, PrPC may play a role in mitochondrial function. In 2019, we continued studies looking at prion disease progression and mitochondrial dysfunction using mice with known mitochondrial defects. In collaboration with Dr. Catharine Bosios laboratory, which has a Seahorse XF Analyzer to measure mitochondrial respiration and viability, we completed studies analyzing mitochondrial function during prion infection in mice in which a gene involved in axonal degeneration has been knocked out. A manuscript based on these data is in progress and should be submitted before the end of the year. We will continue our studies into mitochondrial function and prion disease by infecting mice in which genes that are important in mitophagy have been knocked out. We will then analyze mitochondrial function in these mice in order to determine how mitophagy may influence prion disease progression. In 2019 a new IRTA fellow, Dr. Daniel Shoup, joined the lab and developed cell lines expressing a reporter gene which measures mitochondrial redox stress. We have begun using these cells to determine how PrPC expression and/or prion infection effects the redox state of the cell. The results of these studies will provide insights into the mechanisms involved in cellular loss during prion infection. In 2019, we published a study looking at the fate of host PrPC following intracranial prion inoculation. These studies were designed to provide important information about the events that occur during the first few hours following exposure to prions. We are also in the process of developing a model which uses stereotactic inoculation to target different brain regions to delve more deeply into how PrPC responds to damage to the brain. Amino acid mismatches between PrPC and PrPSc influence the rate at which PrPSc forms. Thus, heterozygosity at key residues has the potential to significantly slow or even prevent disease transmission (J Gen Virol 93: 2749-2756 (2012)). In human PrPC, methionine/valine heterozygosity at codon 129 influences prion transmission and is a known resistance factor to sCJD (ACTA Neuropathol 130: 159-170 (2015)). It is possible that the ratio of PrPSc with methionine at codon 129 to PrPSc with valine at codon 129 (i.e. the PrPSc allotype ratio) determines the efficient transmission of prions in heterozygous cases of CJD. We have previously determined the PrPSc allotype ratio in multiple cases of MV heterozygous sCJD (Moore et al. PLoS Pathog. 12: e1005416 (2016), Annual Report 2016). Last year we completed a long-term in vivo experiment to test the efficiency of transmission of 11 cases of codon 129 heterozygous CJD into two different lines of susceptible mice. In 2019, we finished the biochemical and histological characterization of these mice and expect to publish the results by the end of the year. These experiments will provide important insights into the mechanisms underlying CJD progression in humans and the influence of PrPSc allotype ratio on prion disease tempo and transmission. The prion agent is notoriously difficult to inactivate with the routine sterilization protocols used in hospitals where iatrogenic transmission of CJD is an ongoing concern. The extreme resistance of prions to inactivation and their ability to persist in the environment for decades thus remains a significant public health issue. Similar concerns apply to the laboratory setting where it is often necessary to analyze prion samples using advanced analytical techniques that are frequently only available outside of biosafety level 2 (BSL-2) containment, the minimum biosafety level required for studying infectious prions. However, the remarkable resistance of prions to inactivation can make it difficult to produce and analyze prion samples free of infectivity that still retain sufficient sample integrity for research purposes. In 2018, we published a study demonstrating that a straightforward denaturation and in-gel protease digestion protocol used to prepare prion-infected samples for mass spectroscopy leads to the loss of at least 7 logs of prion infectivity. In 2019, we have expanded these studies to include several other techniques used to prepare samples for mass spectrometry. These experiments will take 1-2yrs to complete but the results will be of use to regulators, biosafety specialists, and researchers tasked with determining whether or not prion-infected samples can be safely analyzed outside of BSL-2 containment.